Blood coagulation is a cascade of hemostatic events during which blood changes from a liquid state to a gel state. Hemostatic events in the body occur only around injured blood vessels, and are elaborately regulated by selective binding between various coagulation factors, proteolysis inhibitors present in plasma, and a series of antagonistic enzyme reactions (Harlan, J. M. et al., Med. Clin. North Am., 65:855, 1981). Despite this elaborate regulation, when the balance between blood coagulation and lysis is broken due to various abnormal or external factors, excessive thrombus formation will occur, resulting in cerebral apoplexy, heart failure and the like (Gormez, C. C. et al., Angiologia, 43:241, 1991).
Cerebral apoplexy is a cerebral nerve disorder, including consciousness disorder and movement disorder, caused by various cerebrovascular diseases, and is a serious disease which is the first leading cause fod death in both men and women in Korea. It is impossible to regenerate tissue necrosis caused by cerebral apoplexy. Thus, to treat the root cause of the disease, risk factors should be removed, treatment for regenerable tissue and the facilitation of regeneration should be conducted first, and mental treatment and physical treatment are also required, because tissue necrosis can be followed by cerebral injury and neurological complications resulting from an increase in cerebral pressure. Methods for treating cerebral apoplexy include providing sufficient blood flow to ischemic areas and using drug therapies, including anticoagulants, anti-platelet agents, thrombolytic agents, blood viscosity-reducing agents, and agent for stopping the progression of ischemia, which protect cells in ischemic changes.
Typical causes of cerebral apoplexy include diseases, such as hypertension, hyperlipidemia, heart disease, thrombosis and diabetes, oral contraceptives, smoking, alcohol drinking and the like. Among these causes, hypertension is the most risky factor that must be treated first (Matlu, N. et al., Arch. Neural., 8:644, 1963), and when problems associated with cerebral apoplexy occur, calcium antagonists such as dihydropyridine and angiotensin conversion enzyme inhibitors are used. Hyperlipidemia causes arteriosclerosis, and thus is treated using diet therapy together with niacin having the effect of suppressing lipolysis, clofibrate and gemfibrozil of having the effect activating lipoprotein lipase to lower tracylglycerol levels, resins binding to bile acid, cholestyramine and colestipol, lovastatin and pravastatin having the effect of suppressing enzyme HMG-CoA reductase to inhibit cholesterol synthesis, and the like.
Meanwhile, anticoagulants are drugs for preventing blood coagulation (thrombosis) caused by either foreign matter (artificial valve) in the cardiovascular system or arrhythmia. Anticoagulant therapy is used for the treatment and prevention of deep vein thrombosis, pulmonary embolism, heart diseases, cerebral embolism, etc., and after artificial heart valve replacement. Typical drugs that are used in such anticoagulant therapy include heparin, a kind of acidic polysaccharide having sulfate groups, which is a strong inhibitor of blood coagulation, and vitamin K antagonist warfarin. In addition, fraxiparine, enoxaparin, etc., are also used. Heparin binds to antithrombin III to show a rapid anticoagulant effect. Antithrombin III is also called heparin coenzyme and inhibits various coagulation factors such as serine protease and thrombin. Because an overdose of heparin reduces rather than increases the activity of antithrombin, thus resulting in the risk of thrombosis, low-dose heparin is used through intravenous injection or subcutaneous injection, and the dose thereof is determined while the degree of blood coagulation in a patient is checked whenever needed.
Vitamin K antagonists include warfarin and dicumarol. Warfarin is an oral anticoagulant showing the effect of inhibiting coagulant factors in the body, and is used in long-term anticoagulant therapy. Warfarin acts instead of vitamin K, a coenzyme involved in the synthesis of proteins involved in blood coagulation, so as to inactivate coagulant factors. Because the inactivation of coagulation factors takes long time, warfarin is not used in acute situations.
Anticoagulants are replaced with other methods about 3 months after the use in cases other than diseases where thrombi are formed. Heparin, a mucopolysaccharide having an anticoagulant effect, is distributed in internal organs having many capillary blood vessels (e.g., liver, lungs, kidneys and muscles) or blood in the higher animals. It is present as a mucoprotein in animal tissues and is purified by extracting it with alkali and removing a protein with an enzyme. It has a molecular weight of about 10,000-20,000 and possesses a structure in which D-glucosamine, D-glucuronic acid and L-iduronic acid are chemically bonded in a chain configuration. It prevents the production of thromboplastin, increases the rate of binding of antithrombin to thrombin to inhibit blood coagulation, and furthermore, purifies blood by inducing and synthesizing lipase that degrades serum lipoproteins. Heparin is a main drug, which is used against venous thrombosis and pulmonary embolism, and reduces the number of thrombosis, stroke. Moreover, it is also used for the prevention of venous thrombosis and acute myocardial infraction after surgical operations. Although it does not pass through the placenta, special attention must be paid to the use thereof in pregnant women.
Meanwhile, fibrinogen is hexaprotein, a kind of water-soluble plasma protein, which has a molecular weight of 340 kDa, is present at a concentration of about 2.5 mg/mL in the body, and is converted into fibrin by thrombin (Craik, C. S. et al., Science, 237:909, 1987). The fibrin thus produced plays an important role in blood coagulation and is a fundamental substance for thrombus formation. Plasmin performs various roles, including the degradation of coagulated fibrin in the body, the inhibition of 2-antiplasmin, the regeneration of injured tissues, and the activation of macrophages (Daka, M. D. et al., J. Vasc. Intern. Radiol., 6:73, 1995). Plasmin is generally present in the form of a zymogen, called plasminogen, and will change into an activated form to show fibrinolytic activity when Arg561-Val562 is degraded by a plasminogen activator (Mullertz, S., Fibrinolysis, 1:3, 1987). This fibrinolytic system has a function of protecting the body from thrombus formation, which is a kind of defense function against the deposition of fibrin in blood vessels. Enzymes having this fibrinolytic activity are mostly serine proteases such as plasmin, and microorganisms such as Bacillus subtilis produce various kinds of serine proteases (Kraut, J., Ann. Rev. Biochem., 46:331, 1977).
Therapeutic agents, which have been used to date for the treatment of thrombosis, include streptokinase, urokinase, tissue-type plasminogen activators (tPA) and the like, but such therapeutic agents employing serine proteases have problems in that they are uneconomical, have a short half life, show side effects such as blood vessel bleeding upon misuse, and are difficult to administer orally, except for urokinase. Recently, the medical college team at Miyazaki University, Japan, has made an oral therapeutic agent by isolating six lumbrikinases from earthworms which have been used for the treatment of palsy, but this product also problems in that it has a short half life and is expensive (Mihara, H. et al., Lubricus rubellus Jan. J. Physiol., 41:461, 1991).
Meanwhile, PGA is a viscous liquid polymer consisting of D,L-glutamic acid bound to γ-glutamyl, and is a natural, edible, water-soluble, anionic, biodegradable polymeric substance, which is produced from Bacillus sp. strains isolated from Chungkookjang (Korean traditional fermented soybean food prepared using rice-straw), Natto (Japanese traditional fermented soybean food) and Kinema (Nepalese traditional fermented soybean food), and can be used as a moisture absorber, a moisturizer and a raw material for cosmetics.
Recently, with respect to the production and utilization of PGA, studies focused on the material of products substituting for non-biodegradable polymers, the development of heat-resistant plastics by esterification and the production of water-soluble fibers and membranes have been actively conducted in, particularly, advanced countries. Also, studies focused on developing and industrializing environment-friendly material, hydrogel having water absorptivity, biodegradability and plasticity, using PGA as a raw material, have been conducted. In such studies, when an aqueous solution containing PGA is treated with a chemical crosslinking agent and irradiated with radiation, the crosslinking reaction between the molecules of PGA can occur to provide PGA resin having water absorptivity, biodegradability and plasticity. Such PGA resin is a polymeric substance, which is highly applicable in hygienic products such as diapers, food, horticultural industries.
Moreover, the following studies and patents, for example, have been reported: the effects of manganese ions on the composition and production of PGA, the use of PGA as water-soluble polymers by untrasonic decomposition, and the development of low-water-soluble plastics by the synthesis of ester derivatives (Ito, Y. et al., Biosci. Biotechnol. Biochem., 60:1239, 1996); the production of PGA by Bacillus subtillis, and the use of PGA as a calcium dissolving agent in healthy foods having an osteoporosis therapeutic effect (JP 6-32742); effects of reducing the content of phosphorus in water systems to reduce water contamination (EP 838160); the use of PGA as biodegradable solid fibers or films and molded films by the dissolution, precipitation and drying of PGA (JP 7-138364 and JP 5-117388); and polymers for drug carriers (JP 6-92870 and JP 6-256220). Thus, PGA is a highly useful natural polymer, which can be used as low-water-soluble plastics, healthy foods having osteoporosis therapeutic effects, agents for reducing water contamination, biodegradable fibers or films and molded films, polymers for drug carriers, and the like.
The present inventors acquired a patent (Korean Patent Registration No. 10-500796) relating to a method of producing poly-gamma-glutamic acid (PGA) using Bacillus subtilis chungkookjang, a halotolerant strain producing high-molecular-weight PGA, and furthermore, acquired patents relating to an anticancer composition containing PGA, an immune adjuvant and an immune booster (Korean Patent Registration Nos. 10-496606; 10-517114; and 10-475406). However, there is still no report on the effects of PGA on anticoagulation and/or thrombus prevention.
Accordingly, the present inventors have made extensive efforts to elucidate the various functions of PGA having various applications and, as a result, found that PGA, which is inexpensive and non-toxic to the human body, has an anticoagulant effect of preventing blood coagulation (thrombosis) caused by arrhythmia, and thus it can prevent thrombi from being accumulated and has excellent water absorption, sustained release, thermal stability, and nontoxic properties to cells, thereby completing the present invention.